Microbiology, 2021

13.2 • Using Physical Methods to Control Microorganisms 517
Figure 13.7
A technician sterilizes a sample using an autoclave. (Credit: Martha Cooper / Picryl; Public Domain.
Standard operating temperatures for autoclaves are 121 °C or, in some cases, 132 °C, typically at a pressure of
15 to 20 pounds per square inch (psi). The length of exposure depends on the volume and nature of material
being sterilized, but it is typically 20 minutes or more, with larger volumes requiring longer exposure times to
ensure sufficient heat transfer to the materials being sterilized. The steam must directly contact the liquids or
dry materials being sterilized, so containers are left loosely closed and instruments are loosely wrapped in
paper or foil. The key to autoclaving is that the temperature must be high enough to kill endospores to achieve
complete sterilization.
Because sterilization is so important to safe medical and laboratory protocols, quality control is essential.
Autoclaves may be equipped with recorders to document the pressures and temperatures achieved during
each run. Additionally, internal indicators of various types should be autoclaved along with the materials to be
sterilized to ensure that the proper sterilization temperature has been reached (Figure 13.8). One common
type of indicator is the use of heat-sensitive autoclave tape, which has white stripes that turn black when the
appropriate temperature is achieved during a successful autoclave run. This type of indicator is relatively
inexpensive and can be used during every run. However, autoclave tape provides no indication of length of
exposure, so it cannot be used as an indicator of sterility. Another type of indicator, a biological indicator spore
test, uses either a strip of paper or a liquid suspension of the endospores of Geobacillus stearothermophilus to
determine whether the endospores are killed by the process. The endospores of the obligate thermophilic
bacterium G. stearothermophilus are the gold standard used for this purpose because of their extreme heat
resistance. Biological spore indicators can also be used to test the effectiveness of other sterilization protocols,
including ethylene oxide, dry heat, formaldehyde, gamma radiation, and hydrogen peroxide plasma
sterilization using either G. stearothermophilus, Bacillus atrophaeus, B. subtilis, or B. pumilus spores. In the
case of validating autoclave function, the endospores are incubated after autoclaving to ensure no viable
endospores remain. Bacterial growth subsequent to endospore germination can be monitored by biological
indicator spore tests that detect acid metabolites or fluorescence produced by enzymes derived from viable G.
stearothermophilus. A third type of autoclave indicator is the Diack tube, a glass ampule containing a
temperature-sensitive pellet that melts at the proper sterilization temperature. Spore strips or Diack tubes are
used periodically to ensure the autoclave is functioning properly.